¹Unité d'Immuno-Hématologie et d'Immunopathologie, Institut Pasteur, Paris, France

Correspondence: Dr B Payelle-Brogard, Unité d'Immuno-Hématologie et d'Immunopathologie, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France. Fax: +33 1 45 68 89 51

Received 11 February 2003; Accepted 13 February 2003.

TO THE EDITOR

Dramatic low expression of the B-cell receptor (BCR), composed of surface immunoglobulin associated with CD79a/CD79b, is characteristic of chronic lymphocytic leukaemia (CLL) B cells.¹ It results in inability to respond to BCR ligation, a state found in anergic B cells.^2,3 Despite normal transcription and intracellular synthesis, we previously showed⁴ that this low expression is accounted for by a defective assembly of the BCR chains resulting in the presence of unprocessed chains. To gain insight into the mechanism accounting for this low expression, we aimed to define the cell compartment where the BCR retention occurs. In addition, as the lack of costimulatory molecules characteristic of CLL B cells can be at least partially restored upon CD40 ligand (CD40L) stimulation,⁵ we investigate the effect of such stimulation on the defective IgM and CD79b intracellular assembly and surface expression.

B cells from four CLL patients and three normal controls were analysed by flow cytometry for surface and intracellular IgM and CD79b expression and by confocal microscopy for the intracellular IgM localization, assembly with CD79b and transport to the cell surface. A CD40L+IL-4 stimulation of CLL B cells was performed, to determine whether it could revert the defective assembly and surface expression. Before stimulation, a consistent low expression of surface IgM and CD79b was observed in CLL patients, which contrasts with normal subjects (Figure 1a). After stimulation (Figure 1b): (1) expression of CD80 and CD86 was significantly enhanced in CLL and normal subjects (for both percentage of positive cells and mean fluorescence intensity); (2) total IgM expression in CLL B cells was moderately upregulated (about two-fold increase in percentage IgM+ cells), however, activated CLL B cells were still expressing poor amounts of surface IgM (mean fluorescence intensity 131 for CLL vs 322 for normal); (3) CD79b surface expression was only detected in 15.2% activated CLL B cells and did not reach normal level, despite a significant increase in CD79b intracellular synthesis (82.9% positive cells after stimulation compared to 27.7% before, data not shown).

Figure 1.

CLL B cells surface expression and CD79b transcripts before and after stimulation with CD40L+IL-4. (a and b) Flow-cytometric analysis of normal or CLL B cells before and after stimulation with CD40L+IL-4. Normal B cells purified through negative depletion or peripheral blood mononuclear cells from CLL patients were stained for IgM, CD79b, CD80, CD86 surface expression, before or after a 6-day culture with CD40L (1 g/ml)+IL-4 (1000 U/ml). The data were reproduced in independent experiments from three normal controls and four CLL patients. The numbers in the upper right corner of each panel are the percentage of IgM+/CD79b+, IgM+/CD80+ or IgM+/CD86+ cells, respectively. (c) RT-PCR analysis of CD79b long (709 bp) and CD79b short (397 bp) transcripts in controls (lanes a–c) and CLL (lanes d–g) before and after CD40L+IL-4 stimulation (indicated by asterisk). The long to short transcripts ratio was calculated in the linear step of the reaction. MW, molecular weight marker.

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Given the key role of CD79b in BCR surface expression, its defective transport could account for low surface IgM expression. CD79b is transcribed in the form of long transcripts coding for the complete protein and short transcripts (CD79b), which are depleted of the extramembrane domain. However, CD79b transcripts could not account for the low surface expression, since the CD79b/ CD79b ratio as assessed by reverse transcriptase polymerase chain reaction (RT-PCR) did not display important differences between normal and CLL B cells before stimulation (0.250.18 and 0.340.06, respectively, Figure 1c) and following stimulation (1.250.16 and 0.690.32, respectively). This is consistent with results reported by Cragg et al⁶ showing that the level of CD79b transcripts does not impair surface BCR expression. Overall, our results demonstrate that CD40L succeeded to induce a moderate upregulation of surface IgM in CLL B cells and almost no change of surface CD79b expression despite an upregulation of costimulatory molecules.

The cellular compartments involved in BCR retention, and the effect of CD40L+IL-4 stimulation on BCR assembly and transport, were then investigated by confocal microscopy analysis after simultaneous staining of IgM and calnexin, a chaperon protein of the endoplasmic reticulum (ER) or golgin, a protein characteristic of the Golgi compartment, or CD79b. While normal B cells showed two separate stainings for IgM (red) and calnexin (green), in CLL B cells IgM colocalized with calnexin as shown by the yellow merged image (Figure 2a); this was still observed after stimulation. As for Golgi compartment, similar colocalization of IgM and golgin was observed for normal and CLL B cells (Figure 2b). However, in contrast to normal B cells, IgM and CD79b did very poorly colocalize (separate red and green stainings) in CLL B cells (Figure 2c), reflecting a defective assembly that was still evident upon stimulation. These results demonstrate an important IgM retention in the ER of CLL B cells, which might play a critical role in defective assembly of the BCR. In conclusion, upon CD40L+IL-4 stimulation costimulatory molecules could be upregulated, although the defective assembly and transport of the BCR could not be reverted. Impairment of glycosylation mechanisms or ER quality control system does not seem to be involved, since glycoproteins like MHC molecules and CD19 are adequately expressed and others like CD80 and CD86 can be upregulated. It remains to be determined whether the BCR retention in the ER in CLL B cells could be accounted for by: (1) an interaction with an as yet uncharacterized intracellular protein as reported for the K1 protein from human herpesvirus 8 in a B-lymphoma cell line⁷ or (2) alterations in IgM motifs leading to defective transport to Golgi as described for CD8 molecule in T cells.⁸

Figure 2.

Confocal microscopy analysis of IgM localization and assembly with CD79b in CLL B cells before and after stimulation with CD40L+IL-4. Peripheral blood mononuclear cells from CLL patients or purified normal B cells, before or after a 6-day culture with CD40L+IL-4, were permeabilized with saponin and colabelled with anti-IgM antibodies (Abs) together with Abs against calnexin (ER compartment, (a), or golgin (Golgi compartment, (b), or CD79b (c). IgM was detected with secondary Ab conjugated with Alexa 546 (red), while CD79b, ER or Golgi was detected with Abs conjugated with FITC, followed by Abs conjugated with Alexa 488 (green). The yellow colour in the merged images indicates colocalization of the IgM with ER or Golgi or CD79b. As identical results were obtained for normal B cells before or after stimulation, only those before stimulation were shown. The data were reproduced in four independent experiments.

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